Murine proinsulin 2 was cloned in frame at the C-terminus of the heavy chain of anti-mCD205 (NLDC-145), which had been previously modified to contain murine Ig κ and IgG1 constant regions with mutations that interfere with Fc receptor binding (21 (link)). The flexible linker (GGGGS)3 was inserted in between the antibody and proinsulin to allow for proper proinsulin folding (20 (link)); this antibody was designated anti-mCD205/Ins2. Anti-mCD205/OVA (15 (link)) and anti-mCD205/NRP-V7 (8 (link)) have been described. The antigen-linked anti-mCD205 antibodies were produced by transient transfection of 293T cells with calcium phosphate and purified as described above for the antigen-linked 3G9 antibodies, except that for each 150 mm plate to be transfected, 30 μg DNA encoding the antibody heavy chain and 30 μg DNA encoding the antibody light chain were used along with 5 μg pAdVantage vector (Promega).
Protocol detail
Antigen-Linked Anti-mCD205 Antibody Production
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293t cells
Anti antibodies
Antibodies
Antibody
Antibody heavy chain
Antibody light chain
Antigen
Calcium phosphate
Fc receptor
Frame
Igg1
Murine
Mutations
Proinsulin
Promega
Transfection
Transient
Vector
Corresponding Organization : Albert Einstein College of Medicine
Other organizations : Celldex Therapeutics (United States)
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Variable analysis
independent variables
- Anti-mCD205 antibody with mutations that interfere with Fc receptor binding
- Insertion of the flexible linker (GGGGS)3 between the antibody and proinsulin
- Proinsulin folding
- Transient transfection of 293T cells with calcium phosphate
- Purification of the antigen-linked anti-mCD205 antibodies
- Anti-mCD205/OVA
- Anti-mCD205/NRP-V7
- Not explicitly mentioned
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